The blocking of capacitative calcium entry by 2-aminoethyl diphenylborate (2-APB) and carboxyamidotriazole (CAI) inhibits proliferation in Hep G2 and Huh-7 human hepatoma cells
Introduction
Calcium plays a key role in cell physiology. The stimulation of liver cells by growth factors results in rapid and sustained increases in intracellular free calcium concentrations, [Ca2+]i [1], [2] leading to the activation of several transcription factors including c-jun, c-myc, and c-fos and of cell cycle-associated cyclin expression [3], [4]. In turn, these transcription factors regulate the expression of several inducible genes mediating diverse programs including cell proliferation, cell differentiation and cell death [5], [6].
Intracellular calcium signals in non-excitable cells, such as liver cells, involve the rapid, transient InsP3-mediated release of calcium from stores, leading to a decrease in the calcium concentration of the ER lumen, followed by the activation of plasma membrane calcium channels to generate a sustained influx of calcium [7]. This type of calcium influx is called capacitative or store-operated calcium entry (CCE) [8]. It is thought to be an essential component of the long-term responses of the cell, including proliferation [9], [10]. Submaximal agonist concentrations unable to trigger intracellular Ca2+ store depletion have been associated with another Ca2+ entry pathway—non-capacitative calcium entry (NCCE)—activated by diacylglycerol [11] or arachidonic acid [12]. It has been known for more than 30 years that the proliferation and growth of normal vertebrate cells are dependent on the physiological concentration of extracellular calcium [13], [14]. Calcium entry is required at various stages of the cell cycle [15], [16] but little is currently known about the nature of the calcium channels involved in this process in non-excitable cells such as liver cells. There is growing evidence to suggest that CCE amplitude is related to cell proliferation in several cell types [10], [17], [18], [19]. The blocking of calcium influx may prevent normal and pathological cell proliferation and there is some evidence to suggest that molecules blocking calcium entry also have antiproliferative properties [20], [21]. Carboxyamidotriazole (CAI), a novel inhibitor of non-voltage-dependent calcium entry, has been shown to have such properties in model systems in vitro and in vivo [16]. In this study, we used two human hepatoma cell lines, Hep G2 and Huh-7, to investigate the relationship between cell proliferation and CCE, using CAI and a known CCE blocker, 2-APB. We suggest that CCE is the main pathway involved in regulation of the processes leading to cell proliferation.
Section snippets
Cell culture
Hep G2 and Huh-7 human hepatoma cells were provided by Dr. Doris Cassio (INSERM U442, Orsay, France). Cells were cultured in complete medium (Dulbecco’s MEM with Glutamax-I supplemented with fetal calf serum (FCS, 10%), penicillin (200,000 U/ml), and streptomycin (100 μg/ml)), at 37 °C, in an atmosphere containing 5% CO2. Hep G2 and Huh-7 cells were plated at densities of 50,000 and 10,000 cells/cm2, respectively, and cultured to about 80% confluence.
Cell proliferation
Hep G2 and Huh-7 cells were plated in 24-well
Calcium measurements
Cell suspensions: Huh-7 cells (5 × 106 cells/ml) were loaded at 37 °C for 45 min with 2 μM Fura-2/AM in complete DMEM culture medium. Cells were treated with trypsin and washed once by centrifugation at 50 × g for 1 min in the same medium. Cell pellets were resuspended in a medium containing 116 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 1 mM NaH2PO4, 5 mM NaHCO3, 0.1 mM EGTA, 20 mM HEPES, pH 7.3, with or without 1.8 mM CaCl2, as indicated. Cell pellets were transferred to a quartz cuvette and placed in the light
Effects of CAI and 2-APB on human hepatoma cell line proliferation
The proliferation of human hepatoma cells was assessed by measuring the incorporation of []thymidine. Cells were first incubated for 24 h in the presence of 10% FCS; the external medium was replaced by fresh medium supplemented with various concentrations of CAI or 2-APB and the cells were incubated for a further 24 h (Fig. 1). CAI blocks the incorporation of []thymidine in Hep G2 and Huh-7 cells, with IC50 of 0.7 and 4.5 μM, respectively (Fig. 1A and B). The incorporation of []thymidine was
Discussion
The overexpression of transient receptor potential (TRP) channels results in increases in CCE and cell proliferation [10], [18], [19], suggesting that these two cellular processes are intimately related. Our results showed that blocking CCE with CAI and 2-APB inhibited proliferation in the Hep G2 and Huh-7 cell lines. 2-APB and CAI inhibit CCE and cell proliferation with similar potency in human hepatoma cell lines, suggesting that sustained increase in [Ca2+]i is required to activate the genes
Acknowledgements
We thank Josiane Simon for technical assistance and Julie Sappa (Alex Edelmann & Associates, Paris, France) for editing the manuscript. This work was supported by grants from Association pour la Recherche contre le Cancer (ARC no. 5241, 5621 and 4615) and Comité de l’Essonne de la Ligue contre le Cancer.
References (46)
- et al.
Cell cycle progression proteins (cyclins), oncogene expression, and signal transduction during the proliferative response of human hepatocytes to hepatocyte growth factor
Hepatology
(1996) - et al.
Discriminating between capacitative and arachidonate-activated Ca2+ entry pathways in HEK293 cells
J. Biol. Chem
(1999) - et al.
Calcium, calmodulin and cell cycle progression
Cell. Signal
(1995) Reduced store-operated Ca2+ currents in rat basophilic leukaemia cells cultured under serum-free conditions
Cell Calcium
(2001)- et al.
Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced Bcl-2 oncoprotein production
J. Biol. Chem
(1993) - et al.
Reciprocal regulation of capacitative and arachidonate-regulated noncapacitative Ca2+ entry pathways
J. Biol. Chem
(2001) - et al.
Calcineurin directs the reciprocal regulation of calcium entry pathways in nonexcitable cells
J. Biol. Chem
(2003) - et al.
Mutual antagonism of calcium entry by capacitative and arachidonic acid-mediated calcium entry pathways
J. Biol. Chem
(2001) - et al.
Smooth muscle cell cycle and proliferation. Relationship between calcium influx and sarco-endoplasmic reticulum Ca2+ATPase regulation
J. Biol. Chem
(1996) - et al.
Hepatocyte growth factor induces calcium mobilization and inositol phosphate production in rat hepatocytes
J. Cell Physiol
(1992)
Epidermal growth factor induces dose-dependent calcium oscillations in single Fura-2-loaded hepatocytes
Hepatology
Activation changes the spectrum but not the diversity of genes expressed by T cells
Proc. Natl. Acad. Sci. U.S.A
Gene regulation mediated by calcium signals in T lymphocytes
Nat. Immunol
Inositol trisphosphate and calcium signalling
Nature
Mechanisms of capacitative calcium entry
J. Cell Sci
Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes
Am. J. Physiol
Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation
Am. J. Physiol
J. Clin. Invest
Calcium as a regulator of the proliferation of normal, but not of transformed, chicken fibroblasts in a plasma-containing medium
Proc. Natl. Acad. Sci. U.S.A
Bringing cell growth research together
Nat. Med
The role of calcium in the regulation of invasion and angiogenesis
In Vivo
Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+ entry in human pulmonary artery endothelial cells
Am. J. Physiol. Lung Cell. Mol. Physiol
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Present address: INSERM U526, Faculté de Médecine Pasteur, Avenue de Valombrose, 06107 Nice Cedex 02, France.